P323: STREAMLINING PRECLINICAL IN VIVO TREATMENT TRIALS BY MULTIPLEXING GENETICALLY LABELLED PDX MODELS OF SEVERAL PATIENTS IN A SINGLE MOUSE
Authors: K. Hunt, D. Amend, R. Ludwig, B. Vick, A. K. Wirth, T. Herold, I. Jeremias
Journal: HemaSphere (2022)

Background: Better treatment options are intensively needed for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Before application in clinical trials, novel therapies require preclinical testing which is resource intensive. In previous work, we had shown that multiplexing several PDX cell populations within a single mouse in competitive in vivo experiments gives identical results to studying each population in separate mice (Liu et al., Biomarker Research 2020). Aims: Here, we aimed at transferring multiplexing to preclinical in vivo treatment trials. We aimed to increase the efficiency of preclinical drug testing in vivo by simultaneously testing several samples in parallel in a single mouse, using a competitive in vivo approach. Methods: From primary patient material, we had established patient derived xenograft (PDX) mouse models of ALL and AML which allow serial transplantation in immunodeficient NSG mice. We designed and cloned 5 different lentiviral constructs to label 5 individual samples with both a unique fluorochrome as well as a connected unique genetic barcode, for later detection by flow cytometry or next generation sequencing, respectively. In addition, luciferase was recombinantly expressed in all PDX models to allow repetitive monitoring of leukemia growth and treatment response using bioluminescence in vivo imaging (BLI). Up to 5 PDX samples, either ALL or AML, were multiplexed and aliquots injected into groups of mice (n=4-6). For post mortem analysis, PDX cells were reisolated from murine bone marrow and spleen and absolute tumor burden of PDX cells was quantified by flow cytometry of exactly 1/10 of the bone marrow and spleen. Determining fluorochrome composition allowed quantifying the proportion that each of the 5 samples contributed to the entire tumor load. In case of treatment, a response rate for each individual samples was determined by comparing cell numbers in treated versus control mice. Results: From our pool of established PDX samples, we selected 5 ALL or 5 AML samples with different molecular alterations, but similar in vivo growth behaviour. Each PDX model was labelled by its individual fluorochrome and genetic barcode, while all models expressed luciferase. Tumor growth as well as the ratio between the samples was repetitively analysed over the entire course of leukemia outgrowth. The composition of the injection mixture was optimized such that each sample accounted for roughly 20% of the total leukemic burden at the time point of start of treatment. Mice containing multiplexed PDX cells from 5 different patients with ALL were treated for 2 weeks with either verum treatment or with solvent as control. In a study using the BCL-2 inhibitor Venetoclax in PDX ALL models, we were able to distinguish between sensitive and resistant PDX samples. While two samples showed a drastic decrease in tumor burden, three samples showed no or only a mild response. This effect could be observed both in cells isolated from bone marrow as well as spleen. Variances between mice were rather small, with few exceptions for some treated mice. With this approach, we were able to define the effect of a single drug on up to five distinct samples in parallel, reducing resources by factor 5. Image:Summary/Conclusion: Taken together, we established a multiplex protocol for in vivo therapy trials that allows simultaneous testing of up to 5 PDX samples in competitive in vivo trials. The approach reduced the required number of experimental mice by a factor 5, in line with the 3R concept. In the future, our approach might rationalize in vivo drug trials.